Topic 7 Recomb DNA repair Flashcards
What are the purposes of homologous recombination
Recombine To create new genetic diversity by exchanging genetic information between two dna molecules with similar sequences
Recombination repair To fix singles strand and double strand breaks
How is meiosis both helpful and not helpful
Meiosis gives genetic exchange and diversity
but to do this chromosome arms need to break which is Dangerous because if the breakage doesn’t properly repair, loss of dna info and then mutations can happen
How do DSB arise
During DNA replication when the fork encounters a ss break in the template strand (now goes into the daughter strand)
Meiotic recombination
Exposure to UV light or gamma radiation
Oxidative DNA damage during respiration
Explain what scenarios happen when there is a damaged template strand during replication
- Translesion synthesis: just read and moves past the lesions
- The replication fork stalls at the lesion causing fork regression
- If a full single stranded break in the template, fork collapses, and a double stranded break is formed with the template strand (other strand is normal length)
- The lesion is bypassed/jumped over, which leaves a single stranded gap in the daughter strand, replication continues downstream
Expo n what happens right after a double stranded dna break happens (in one chormosomes)
Exonucleases make 3’ overhangs by chewing the 5’ ends of the broken dna stands
DNA recombinase causes one 3’ overhang strand to invade and recombine with the homologous chromosome, making a D-loop structure (due to displacement of the homologous strand during invasion)
Double crossover: The D-loop migrates/expands and allows the other end of the broken strand to also invade (2nd strand invasion)
The bottom broken strand uses the bottom homologous strand as a template and the top broken strand uses the top homologous stand as a template
The 3 prime overhangs are used as primers for dna pol to extend and repair the broken strands using the homologous strands as a template
What is used as a primer in DSBR to extend the broken strands
The 3’ overhangs are used as primers for extension
What are the two ways to complete DSB repair
SDSA (synthesis dependent strand annealing) pathway
DSB repair pathway
What is SDSA (synthesis dependent strand annealing) pathway
After using the homologous chromosome as a template, the invading strands dissociates and anneal to each other and ligate
No crossing over event, just repairing the lesions using the homologous as a template
What is DSB repair pathway
The invading strand is linked in the d loop structure
This makes 2 Holliday junctions/intermediates that are fixed by Holliday intermediate resolvases
Can be resolved in two ways
What are the two ways to resolve the Holliday junctions and the out comes
X x X resolution: non crossover
X x Y resolution: cross over and exhange of genetic info
Explain what happens during a fork collapse
When there is a break in the template strand in the replication fork
The lagging (top) strand keeps going but the leading strand stops at the break and the arm with the break detaches
causing a DS dna break and The fork collapses
the repair needs a reattachment of the broken arm to recreate the replication fork
Explain how recombinational DNA repair fixes a collapsed fork
Have the bottom normal chromosme and the dS break top chromsome
Exonucleases remove part of the 5’ end of the broken strand (so cleave the 3-5 stand) to make a 3’ overhang on the bottom strand (5-3 strand)
Recombinase binds to the 3’ overhang to promote strand invasion and make a Holliday junction
The 3’ overhang pairs with its complimentary strand on the homologous chromsome
Then branch migration occurs to let the strand invade further
Fork is remade when the Holliday intermediate is resolved followed by ligation
Whag is branch migration
The movement of a branch point in the branched DNA formed by two DNA molcules with identical sequences
This can make a Holliday junctions but The net amount of duplex dna does not change
Explain what fork regression is
When a lesion is encountered in the replication fork, the fork starts to move backward
The fork was open then starts sealing back up
This lets the lesion stay reannealed with the template strand (not replicated since not in the fork)
Explain the steps of fork regression
Fork Encounter lesion
The replicated strands in the fork go in the opposite direction (backward) so the fork stays closed
NER can repair the lesion and restart replication, the extra regressed replicated strands (short dna arm) get digested
Or
No NER, the short dna arm replicates using the other regressed strand as a template
Then the replication fork opens more to let the newly synthesized strand pair with the lesion (not perfect pairing) so replication can restart
The lesion can be repaired later
Overall What does fork regression do
Restarts replication
When is dna replicated
Before meiosis
Whag are the stages of prophase 1 in meiosis and what do they mean
Leptotene: the chromosomal condensation happens
Zygotene: the homologous chromosomes cross over through synapsis making the synaptonemal complex to allow recombination
The synapsed chromosomes make a bivalent/tetrad
Pachytene: synapsis is conpleted
Diplotene: synaptionemal complex disappears and homologous chromosomes start to move apart
What are chiasmata
Remaining points of attachment between homologous chromosomes during crossover/exchange
Which histone can you use to scan the chismata during exchange
When dna is breaking during recombinations
you can see h2A-X because it replaces H2A at the site of double stranded breaks
What needs to happen for meiotic recombinations to happen
double stranded breaks in The chromsome
What catalyzes double stranded breaks during recombination and how
Spo11 in S.Cerevisiae
Uses its tyr residues (has oh) to act as a nucleophile in a transesterification reaction
This made the DSB + spo11 with spo11 linked to the DSB by a 5’ phosphotyrosyl linkage
The are 2 spo11 one on top strand one on bottom strand
What is spo11 related to
It is closely related to the Topo II in eukaryotes
Explain what happens after spo11 male the DSB to prepare for recombinations
The Mre11-Rad50-Xrs2 endonuclease complex binds to each spo11 and cleaves the dna on the 3’ side of each spo11 to release the 5’ ending dna fragment with spo 11 attached
This makes 3’ overhangs
Nuclease Sae2 degrades the 5’ end of cleaved strand more to enhance the 3’ overhangs
Sgs2-Dna2/exo1 complex further degrades the 5’ ends to make even longer overhangs
Then RPA is loaded into the 3’ overhangs to keep the dna single stranded and RecA recombinases are allow for strand invasion for recombination
What are the two possible fates of DSB in meiosis
Crossover or non crossover
But both have gene conversion
What is gene conversion
The non reciprocal transfer of genetic info (meaning other strand doesn’t get changed) as a result of DNA repair especially during meiosis
How can recombination happen during mitosis
Does it happen often
Rare
Caused by Exposure to ionizing radiation, endonucleolytic action, a replication fork with a template strand break
When does mitotic recombination happen
What happens after the DSB happens
Not during mitosis
During the S and G2 phases of cell cycle
The DSB triggers a checkpoint that halts the progression of the cell cycle to allow repair to occur
How does mitotic DSB repair happen
Similar to meiotic recombination
Happens like this:
A terminally differentiated cell has a Mitotic DSB
Nuclease Make 3’ overhangs
RPA coats the single stranded dna to keep single stranded
Rad52 and Brca2 recombination mediator protiens come
Rad52 recruits Rad51 and they both start recombinations, rad51 triggers the first strand invasion and rad 52 triggers the second
Leads to either SDSA or DSBR
Other than using DSB to recombine and repair what do DSB help with
Switching between different mating types
What two mating types does the haploid state of S .cerevisiae have
The S cerevisiae can be in diploid or haploid state
Would prefer diploid state but when conditions are not good they go into haploid state
In haploid state: a or alpha (like male or female)
The genes in the MAT locus in their chromosome determines whether they are a or alpha
Explain how the mating types determination in haploid S cerevisiae works
Have the chromosme with
HMLaplha, mat locus, and HMRa
The genetic info for MAT alpha is stored but not expressed in HMLaplha
The genetic info for MATa is stored but not expressed in HMRa
The only time aplha or a is expressed is if those MAT genes are in the MAT locus
Explain how recombinational repair of DSB switches the mating type in haploid S cerevisiae
50/50 chance of cells expressing a or aplha mating types, meaning they switch across generations
The nuclease HO cleaves the mat locus to make a DSB
The free ends are processed to make 3’ overhangs
RAD51 binds to the overhangs to do strand invasion into the new mating type chromosme
Nucleases remove the original mating type info
DNA pol extends the invading 3’ end using the new mating type chromosme as a template
Depending on what mating type was expressed in the mat locus before the break, the opposite type will come in to fix the break through gene conversion
Ex. MATa broke, recombines with HMLaplha comes, now MATalpha
This is done by SDSA repair
How can DSB be repaired if the homologous template strands aren’t available
Non homologous ends joining (NHEJ)
When does NHEJ happen
The differentiated mammalian cells that rarely divide spend most of their time in the G1 and G0 phases, they use NHEJ to repair their dna damages
But NHEJ doesn’t not conserve the original DNA sequences meaning it can introduce mutations
Explain how NHEJ happens
The DSB happens in G1 or G0
The ku70 and ku80 heterodimer binds to the broken ends of the DNA strands and act as molecular scaffolds
They recruit DNA-PK (kinase) and the nuclease Artemis
This ku/DNAPK/Atermis complex hold together (synapse) the broken ends of the DNA
helicase separates the DNA which activates the DNA PK
DNAPK phosphorylates Artemis to activate its endonuclease activity
Artemis cleaves the DNA segments to turn the previous blunt ends into sticky ends
Then the strands are annealed, the small DNA gaps are filled by polymerases
The nicks are sealed by a protien complex called XRCC4, XLF, and DNA ligase IV
What causes there to be more mutations in NHEJ
Since the Artemis endonuclease cleaves the blurb ends to make sticky ends then these ends are just stuck together
some dna info is lost when this happens and sequence is lost and mutation can happen
But choose between survival and death
What are XRCC4 and XLF
X ray cross complementation group
XRCC4 like factor
How can DNA repair systems be used for gene editing
In CRISPR, we can knock in or knock out a tag
HOMOLOGOUS RECOMBINATION: To knock in this tag (ex GFP) we can use a designed donor dna with the tag to recombine with the target dna that has a DSB from the CAS9 from the crispr
NHEJ: to knock out a gene (make mutations) the DSB in the DNA from Cas9 can undergo NHEJ since no template. A mutation is introduced and the gene is non functional
